Production of extracellular invertase by certain strains of yeasts



PRODUCTION or EXTRACELLULAR INVERTASE BY CERTAIN STRAINS OF Robert G. Dwors chack and Lynferd J.' Wickerhalny Peoria, 111., 'assignors' to the United States-'of'America of 1 percent additional sucroseand omission of the agar. The medium was adjustedto pH 6.0 with sulfuric acid before sterilization. The Erlenmeyer flasks were stoppered with cotton plugs and the Fernbach flasks were as represented by the Secretary'ofAgriculture N Drawing.

' Z-Claims. (Cl-f19 5-- '65)1Q j (Granted under Title- 35, U.S. Code 1952 see. 2561 7 ,A non-exclusive, irrevocable,royalty-free licenseini were incubated for 24 to 48 hours at 28 C., the in all purposes ofthe United States, Government, with the power to grant sublicenses for such purposes, is hereby.

granted to the Government of'the United States of America. l

This invention relates to the preparation of an enzyme product having high invertase activity for certain yeasts known as Saccharomyces, Candida, Saccharomycodes, Schizosaccharomyces, Zygosaccharomyces, Endomycopsis, Torulaspora, Kluyveromyces and Debaryomyces.

The invertase enzyme product is prepared bypropagating yeast of the species known to produce considerable amounts of extracellular invertase in a liquid medium, separating the yeast cells from the clear broth, and evaporating the broth to obtain the invertase preparation in the form of a sirup or solid. 1 I

. Invertase is used to produce non-crystallizable invert sugars from sucrose for the food and baking industries. The enzyme is used in the confectionery industry to make soft centers in chocolate coated candies, and by the paper industry to make levulose for use as a plasticizing agent. Invertase is also used in the preparation of D-fructose from inulin, meliboise from raflinose,; gentiobiose from gentianose, and phosphorylated hexose produce an adequate amount of invertase to hydrolyze the sucrose in the molasses.

The advantages of the invertase products of our invention over the invertase products of the prior art in cubation temperatureemployed in all experimental work reported here. The composition of the liquid medium used to prepare inoculum, as well as invertase solutions, are similar to'that of the agar medium with the exception capped with 6 milk filter pads each. All flasks were sterilized by 'autoclaving at 121 C. for 25 minutes.

.The liquid'inoculum was'prepared' by washing the" cells of a slantculture into 5 m1. of sterile water and transferring 1 ml. of the suspension into a 500 ml. Erlenmeyer flask containing 100 ml. of medium. to 48 hours incubation, 25 ml. were used to inoculate 5-00, ml. of medium-in Fernbach flasks which were used in all of the laboratorystudies and for the subsequent inoculation of 'ZO-liter stainless steel, fermentors. The flasks were incubated on a reciprocating shaker making ninety 3-inch cycles per minute. A modification of the Sumner and Howell method (J. Biol. Chem., 108:51,

added to stop further hydrolysis. The number of mgs'.

of invert sugar formed in 1 hour at 20 C. and pH 4.5 expresses directly the invertase activity of one milliliter of culture broth.

The buffer solution was prepared by dissolving 5.85 g. of sodium acetate trihydrate and 57 ml. of N acetic acid in distilled water and diluting to one liter. The

- sucrose-butter solution was prepared by dissolving 6.5 g.

which the invertase products are prepared by the autolysis of yeast cells, are their greater purity, greatly reduced adulteration by other enzymes and proteins, and freedom particularly from melibiase. An additional advantage is that extracellular invertase preparations may be produced from the effiuent liquors of a food or feed yeast plant.

This invention demonstrates that a number of sucrosefermenting yeasts produce extracellular invertase. Table III, column 4, contains data on the production of this The yeast cells are;

counts.

' following laboratory study was made. Five hundred milliliters of medium in each of two -Fernbach flasks was inoculated and incubated under aerobic culture conditions as previously described. Fourteen hours after inoculation growth was abundant and samples were withdrawn for assay of invertase activity and for live and dead cell Twenty-five milliliters of toluene was added to one flask and the cotton enclosure was replaced by a then removed by centrifugation-and the clear broth is eminently suitable for use as a source of the invertase" enzyme. The invention is further demonstrated by the following examples. a 1

EXAMPLE 1 The yeast, Sacchammycesuvarum NRRL Y-972, was

taken from lyophil culture and transferred daily on an The broth may be concentrated by rubber stopper to prevent evaporation of the toluene. A second and similar culture received no toluene. Both were again placed on the shaker and aliquots of each were withdrawn at intervals and assayed. The data are given in Table I. It will be noted that the culture without toluene contained relatively few dead cells but instead many live and actively growing cells which produced extracellular invertase at a rapid rate throughout the first 90 hours. However in the other flask, the rapid rise in extracellular invertase stopped shortly after the yeast cells were killed with toluene.

. ing 186 hours of the experiment. In addition, 26 percent agar medium composed of 2 percent-sucrose, 0.3 percent yeast extract, and 0.5 percent peptone. The slants of the yeast cells were autolyzed, as represented by the decrease-in cell counts, but the invertase activity in the filtrate changed very little. This, it is shown that enzyme activity increased as the number of actively fermenting live yeast cells increased.

After 24 I 2,953,500 3 4 Table la -Secretion of invertase by living and dead yeast EXAMPLE 3 'Y"972 under anaerobic culture conditions Unisexual forms of Saccharomyces kluyveri and closely Treated Culture related species may be produced by streaking the haploid (Toluene Added) mating types on YM agar plates. When. the well-isolated Untreated Culture Time, colonies areapproximately 17 days-oldsecondary'oolonies hrs. Invertase Invertase "Live Yeast Dead. Yeast Activlty, Dead Yeast Activity, appear on the surface ofithepnmary colonies. When the Cells X106 Ceufixw X106 cultures are. 20 to 30 days old, the secondary colonies are i of two colors, white and brown. The white colonies are 312.3 gg generally unisexual diploids or tetraploids, and the brown 30010 g 1 :5 1 1 variants are believed to be mainly unisexual triploids.

3. gig i 2%; The secondary colonies are restreaked for purity. The

7 3- i brown isolates are commonly unstable, but may be stabilized by the fiollowing. procedure. llsollates are inoculated at three equidistant points forming the tips of atriangle on the surface of each of two petri dishes using a sharp, bent needle. The plates are incubated for 20 to 30 days at C. Cells are taken from the outside edge of a sector which is of the same appearance and continuous with the growth occurring at the center of one of the six colonies. The cells 25 are used to inoculate a second set of plates. Mature colonies of the second or third serial plating are usually homogeneous in appearance, thus indicating stability. Such cultures may be lyophilized to maintain stable cultures indefinitely. For illustrative purposes, Saccharo- Enzyme control in presence of toluene: Activity of filtrate, 58.9 units/1111.; activity of filtrate treated with toluene for 200 hrs, 59.1 units/ml.

EXAMPLE 2 One-liter Erlenmeyer flasks containing 200 milliliter of medium, were inoculated as described in Example 1. The flask was incubated in still culture under anaerobic conditions. Samples were withdrawn from time to time and assayed for invertase activity. The data are given in Table I I.

Table lI.-Secretion of invertase. by S. uvarum NRRL cells. under aerobic culture conditions myces kluyveri Code 3 was grown in 200 milliliter Invertase medium contained in a 1-liter Erlenmeyer flask under Live Yeast Dead Yeast Activity in Time, Hm Cells, Cans Fmmt' the culture conditions cited 1n Example 1. Samples were gwithdrawn at varying mtervals and assayed for mvertase activity. The data showed that 1310.0 and 1906.0 units 31 of invertase were elaborated in the medium in 1 14 and 33-3 f}; 163 hours, respectively. These figures are very much higher than for any other type of yeast studied.

.Table- III.-Y'east species whichv produce extracellular invertase Invertase Activity; units/ml. Name of Culture Number I Aerobic Anaerobic Saccharamyces kZm z/erl (unlsexual haploid) Y-4288-7H1 Dn Y-4288-l3H2 Saccharomyces kluyveri (unisexual diploid) Y-4288 7D2 Dn. Y-428813D2 Saccharomyces kluuven' (unlsexual polyplold) Y-4288-Code 3 Dn Y-4288-C0de 4 491. 0 267. 2 D0 380 5 223 6 D Dn Do Do Saccharomyces klm ven' (bisexual diplold) Saccham'myces kluyverz (bisexual triploid) Saccharomyces kluyvm' (bisexual tetraplold). Candida (Torzllopsis) mills- Candida (Torulopsis) ulilis v. thermophilm Candida (Toru lopais) utilia v. major Tortllopsl; colhculosa... 210. 8 7.1 Saccharomz cea uramm 138.0 51.0 Sacchammuces validus 53. 9 26.0 Saccharomyces fragilis 38.3 8.3 Sacchammz ces om'formla 36.0 20. 8 Saccharomycas cercvlsiae..- 31. 6 45. 4 Saccllaromyces lactis 27. 7 30. 7 Saccharomyces carlaberaemis 5. 7 4. 0 Zygosaccharomuces ashbvi--. 84. 0 63. 4 Kluyueromyces pol spams 54. 6 54. 2 Torulaspora rosei 50. 6 7. l Schizosaccharomyces japonlc'ua 41. 3 38. 6 Sascha, Zudw g 14.0 16. 7 EndomycopsischodalL 13. 1 3. 5 E? 's, 2.9 26.8 Candida flaren'. Y1476.. 5. 4 15. 4 Debaryomyces kloeckerl. Y833 0 20. 5

Incubation temperature: 28' G. Incuba ion i e: 144 hours.

EXAMPLE 4 The production of extracellular invertase in 20-liter' stainless steel fermentors was studied. The description and operation of the fermentors are described by Dworschack, Lagoda and Jackson (Applied Microbiology, 2: 190-197 (1954)). One of the media used in the fermentors consisted of 3 percent sucrose, 0.3 percent yeast extract, and 0.2 percent acid hydrolyzed wheat gluten. The medium was sterilized for minutes at 130 C. Inoculum for the fermentations was prepared as described in Example 1. A total of 10 liters of inoculated medium was used in each fermentor. The culture conditions consisted of a temperature of 28 C., an agitator rate of 200 rpm, and an aeration rate of 0.25 volume of air/volume or medium/minute without head pressure. The fermentors each contained 4 bafiles and one stone sparger. Two and one-half milliliters of silicone antifoam were added at the beginning of the fermentation and once again 16.5 hours later to prevent excessive foaming.

The progress of the fermentation 'was followed by withdrawing small samples at varying times for invertase analysis. The results showed that 91.6 units of extracellular invertase were produced during 97 hours. These results compare favorably with those obtained from the laboratory studies.

EXAMPLE 5 Since invertase is a constitutive enzyme, it is important to determine the amount of extracellular invertase a yeast will produce in media containing a carbohydrate other than sucrose. An industrially-important yeast, Candida (Torulopsis) utilis and varieties major and thermophila were selected for study. This species and its varieties are used for the production of food and feed yeast from molasses especially in tropical countries, and from glucose and pentose containing suliite waste liquors in temperate countries. The conversion of industrial wastes into useful products by fermentation is becoming increasingly important. The recovery of invertase from the fermented efliuents would make these processes more profitable.

For illustrative purposes, the culture, Candida utilis,"

NRRL Y-900, commonly used in sulfite liquor, and Y- 1082 and Y-1084, commonly used in molasses, were used in this study. The yeasts were taken from lyophil culture and transferred twice at 24-hour intervals on an agar medium containing l-percent glucose or l-percent sucrose, 0.3-percent yeast extract, 0.3-percent malt extract, and

0.5-percent peptone. One hundred ml. of medium containing 3-percent glucose or 3-percent sucrose, 0.3-per- Table I V.Eflect of glucose on production of extracel-i lular invertase by certain yeasts grown in aerobic culture Invertase activity Name of culture N o.

Glucose Sucrose Candida (Torulopsis) utills Y-900 369.0 354.0 Candida (Torulopsis) utilis v. thermophila Y-1082 178.0 170.0 Candida (Torulopsis) utilis v. major Y-1084 131.0 113.0

We claim:

1. The process of preparing concentrated extracellular invertase compositions which comprises the cultivation of a species of yeast selected from the group consisting of Saccharomyces kluyveri (uuisexual forms) Candida (T orulopsis) utilis and varieties major and thermophila, Torulopsis 'colliculosa, Saccharomyces uvarum, Saccharomyces validus, Saccharomyces fragilis, Saccharomyces oviformis, Saccharomyces lactis, Saccharomyces carlsbergem sis, Zygosaccharomyces as'hbyi, Kluyveromyces polysporus, Schizosaccharomyces japonicus, Torulaspora rosei, Saccharomycodes ludwigii, Endomycopsis fibuliger, Endo'mycopsis chodati, Candida flareri, and Debaryomyces kloeckeri in a carbohydrate medium containing assimilable nitrogen, separating the unautolyzed yeast cells from the broth and preparing from the cell-free broth an enzyme composition with the desired content of invertase.

2. The process of claim 1 in which the yeast species is cultivated in a carbohydrate medium selected from a group consisting of sucrose, glucose and maltose.

UNITED STATES PATENT OFFICE CERTIFICATE OF CORRECTION Patent No. 2,953,500 September 20, 196d Robert G. Dworschack et al.

It is herebfi certified that error appears in the-printed specification 1 of the above numbered patent requiring correction and that the said Letters Patent should read as corrected below.

Column 1, line 34, for "meliboise" read melibiose column 2, line 71, for "This" read Thus column 3, 'Lgjfible in the title thereto, line 2, for "Y-972 under anaerobic 'read cells under aerobic same column, Table II, in the title g thereto, line 1, after "NRRL" insert Y-972 line 2 of the title, strike out "cells"; same line, for "aerobic" read anaerobic columns 3 and 4, Table III, first column there 0 under the heading "Name of Culture", line 19, for "Saccharomyce urarum" read Saccharomyces uvarum Signed and sealed this 11th day of April 1961. E (SEAL) 1 Attest:

EB??? 1. .S Y ARTHUR w. CROCKER Attesting Oflicer Acting Commissioner of Patents 

1. THE PROCESS OF PREPARING CONCENTRATED EXTRACELLULAR INVERTASE COMPOSITIONS WHICH COMPRISES THE CULTIVATION OF A SPECIES OF YEAST SELECTED FROM THE GROUP CONSISTING OF SACCHAROMYCES KLUYVERI (UNISEXUAL FORMS) CANDIDA (TORULOPSIS) UTILIS AND VARIETIES MAJOR AND THERMOPHILA, TORULOPSIS COLLICULOSA, SACCHAROMYCES UVARUM, SACCHAROMYCES VALIDUS, SACCHAROMYES FRAGILIS, SACCHAROMYES OVIFORMIS, SACCHAROMYCES LACTIS, SACCHAROMYCES CARLSBERGENSIS, ZYGOSACCHAROMYCES ASHBYI, KLUYVEROMYCES POLYSPORUS, SCHIZOSACCHAROMYCES JAPONICUS, TORULASPORA ROSEI, SACCHAROMYCODES LUDWIGII, ENDOMYCOPSIS FIBULIGER, ENDOMYCOPSIS CHODATI, CANDIDA FLARERI, AND DEBARYOMYCES KLOECKERI IN A CARBOHYDRATE MEDIUM CONTAINING ASSIMILABLE NITROGEN, SEPARATING THE UNAUTOLYZED YEAST CELLS FROM THE BROTH AND PREPARING FROM THE CELL-FREE BROTH AN ENZYME COMPOSITION WITH THE DESIRED CONTENT OF INVERTASE. 